High affinity binding site of HGFR and methods for identification of antagonists thereof

ABSTRACT

Use of a polynucleotide encoding or a polypeptide comprising at least the extra-cellular IPT-3 and IPT-4 domains of hepatocyte growth factor receptor for the screening and/or development of pharmacologically active agents useful in the treatment of cancer, preferably a cancer with dysregulation of hepatocyte growth factor receptor.

This application is a divisional of U.S. application Ser. No.12/465,307, filed May 13, 2009, which claims priority to EuropeanApplication No. 08103958.8, filed May 14, 2008, the entire contents ofwhich are hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the area of the hepatocyte growthfactor receptor (HGFR) protein. More specifically, the present inventionrelates to the identification of the high affinity binding site of HGFRfor its ligand, the hepatocyte growth factor (HGF), and methods for theidentification of antagonists of HGFR targeting the high affinitybinding site of HGFR.

BACKGROUND OF THE INVENTION

The hepatocyte growth factor receptor (also known as Met) is a tyrosinekinase and is the product of the c-met proto-oncogene. It consists of a50 kDa α-subunit and of a 145 kDa β-subunit, which are linked by adisulfide bond, the α-subunit being completely extracellular, while theβ-subunit includes (from N- to C-terminus) an extracellular region, atransmembrane domain and a cytoplasmic tyrosine kinase domain. Themature α/β hetero-dimeric receptor is generated by proteolyticprocessing and terminal glycosilation from a 170 kDa single-chainprecursor.

HGF, also known as Scatter Factor, is a heparin-binding glycoproteinwith a broad spectrum of biological activities including cellproliferation, motility, survival and morphogenesis. It is synthesizedand secreted as an inactive single chain precursor (pro-HGF) that isstored into the extracellular matrix due to its high affinity forproteoglycans. Pro-HGF undergoes proteolytic cleavage at residuesR494-V495 to give rise to the biologically active form, adisulfide-linked α/β hetero-dimer, where the α-chain consists of anN-terminal domain followed by four kringle domains and the β-chainshares structural homology with the chymotrypsin family of serineproteases. The β-chain, however, lacks proteolytic activity since two ofthe three critical residues that form the catalytic triad typical ofserine proteases are not conserved in HGF. Despite its inability tosignal, pro-HGF binds to Met at high affinity and displaces active HGF.

Recently, a number of structure-function studies have shed some lightonto the interactions between the extracellular portion of Met and HGF.

The Met extracellular region has a modular structure, which encompassesthree functional domains: the Sema domain (present also in Semaphorinsand plexins) which spans the first 500 residues at the N-terminus of theprotein and has a seven-bladed β-propeller structure, the PSI domain(also found in Plexins, Semaphorins and Integrins) which covers about 50residues and contains four conserved disulfide bonds, and additional 400residues which link the PSI domain to the trans-membrane helix and areoccupied by four IPT domains (Immunoglobulin-related domains present inPlexins and Transcription factors).

HGF is a bivalent ligand, containing a high affinity binding site forMet in the α-chain and a low affinity binding site in the β-chain.Cooperation between the α- and the β-chain is required for thebiological activity of HGF; while the α-chain, and more precisely theN-domain and the first kringle, is sufficient for Met binding, theβ-chain is necessary for Met activation.

Resolution of the crystal structure of the SEMA and PSI domains of Metin complex with the β-chain of HGF (see i.a. WO-A-2005/108424) revealedthat the low affinity binding site for HGF is located in blades 2-3 ofthe β-propeller, and that the portion of HGF-β that binds to Met is thesame region that serine proteases use to bind to their substrates orinhibitors. Importantly, determination of HGF β-chain crystal structureat 2.53 Å resolution and specific mutagenesis analysis unveiled that theresidues involved in Met binding in the activation pocket of HGF β-chainget exposed only following proteolytic conversion of pro-HGF, thusexplaining why pro-HGF binds to Met at high affinity without activatingit. While the low affinity interaction between the β-chain of HGF andthe Sema domain of Met is well characterized both structurally andfunctionally, at the moment it is not clear what region of Met binds tothe α-chain of HGF at high affinity. Thus, the main mechanism by whichHGF activates Met still remains poorly understood. This is somehowsurprising when considering the great biological and therapeuticimportance of this pathway.

HGF-Met signaling is essential during embryogenesis and in tissueregeneration in the adult life. Importantly, deregulated HGF-Metsignaling plays a key role in tumorigenesis and metastasis.Inappropriate Met activation by different mechanisms including autocrineHGF stimulation, receptor overexpression, gene amplification and pointmutation is described in a wide variety of human malignancies andcorrelates with poor prognosis. These findings resulted in a growinginterest in the HGF-Met pathway as a target for cancer therapy, leadingto the development of a variety of Met/HGF inhibitors. These includesmall molecule compounds targeting Met kinase activity, neutralizinganti-Met or anti-HGF antibodies, decoy receptors and HGF-derivedfactors. Nevertheless, whether such molecules target the high affinitybinding site for HGF and which is the exact molecular mechanism at thebasis of HGF binding to Met at high affinity, remains still unknown.This may prevent from isolating more selective therapeutic agents, withincreased sensitivity and fewer side effects. The exact knowledge of theMet high affinity binding site for HGF will certainly help to designhighly specific antagonists of Met.

SUMMARY OF THE INVENTION

The need is therefore felt for improved solutions enabling theidentification of Met antagonists with increased sensitivity and fewerside effects for the development of more effective therapeuticstrategies for the treatment of cancers.

The object of this disclosure is providing such improved solutions.

According to the present invention such objects are achieved thanks tothe solution having the characteristics referred to specifically in theensuing claims. The claims form integral part of the technical teachingherein provided in relation to the present invention.

Thus, object of the present disclosure is the identification of the HGFhigh affinity binding site to the hepatocyte growth factor receptor(HGFR), i.e. the extracellular IPT-3 and IPT-4 domains of HGFR. Afurther object of the present invention is to provide means for theidentification of antagonists of HGFR targeting the high affinity bidingsite of HGFR for HGF for the development of new therapeutic strategiesfor the treatment of cancers.

In an embodiment, the present invention provides for the use of apolypeptide comprising, or a polynucleotide encoding at least theextracellular IPT-3 and IPT-4 domains of hepatocyte growth factorreceptor for the screening and/or development of pharmacologicallyactive agents useful in the treatment of cancer, in particular a cancerwith dysregulation of hepatocyte growth factor receptor activity.

In an embodiment, the pharmacologically active agent is a hepatocytegrowth factor receptor inhibitor and/or antagonist and can be selectedamong small molecule inhibitors, aptamers, antisense nucleotides,RNA-based inhibitors, siRNAs, antibodies, peptides, dominant negativefactors.

In a further embodiment, the present invention concerns a method todetect the ability of a test agent to act as an antagonist/inhibitor ofhepatocyte growth factor receptor useful in the treatment of cancer,preferably a cancer with dysregulation of hepatocyte growth factorreceptor activity, comprising the steps of:

(a) putting in contact a test agent with at least the extracellularIPT-3 and IPT-4 domains of hepatocyte growth factor receptor, or cellsexpressing at least the extracellular IPT-3 and IPT-4 domains ofhepatocyte growth factor receptor,

(b) measuring hepatocyte growth factor receptor activity, function,stability and/or expression, and

(c) selecting the agent that reduces hepatocyte growth factor receptoractivity, function, stability and/or expression.

In a still further embodiment, the present invention concerns the use ofthe extracellular IPT-3 and IPT-4 domains of hepatocyte growth factorreceptor as a medicament for treating cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail in relation tosome preferred embodiments by way of non-limiting examples withreference to the annexed drawings, wherein:

FIG. 1 shows the engineering and purification of Met and HGF subdomains.(A) Schematic representation of the engineered proteins used in thisstudy. Left panel: engineered receptors. W.T. MET, wild-type Met; EXTRA,extracellular portion; INTRA, intracellular portion; SP, signal peptide;SEMA, semaphorin homology domain; PSI, plexin-semaphorin-integrinhomology domain; IPT 1-4, immunoglobulin-plexin-transcription factorhomology domain 1-4; TM, trans-membrane domain; JM, juxta-membranedomain; KD, kinase domain; CT, C-terminal tail; E, FLAG or MYC epitope;H, poly-histidine tag. The red triangle indicates the proteolyticcleavage site between the α- and β-chain. Right panel: engineeredligands. W.T. HGF, wild-type HGF; ND, N-domain; K 1-4, kringle 1-4; PLD,protease-like domain; UNCL. HGF, Uncleavable HGF. The asterisk indicatesthe R494Q amino acid substitution in the proteolytic site. (B) Coomassiestaining of affinity-purified receptors and ligands. Each protein group(Sema, Sema-PSI, Decoy Met; PSI-IPT, IPT; HGF-α, Uncleavable HGF, HGF;HGF NK1, HGF-β) has been resolved by SDS-PAGE in non-reducing conditionsand is quantified against a standard curve of bovine serum albumin(BSA). MW, molecular weight marker; kDa, kilo-Dalton.

FIG. 2 shows an ELISA analysis of HGF-Met interactions. (A) Binding ofMet sub-domains to active HGF. Engineered receptors were immobilized insolid phase and exposed to increasing concentrations of active HGF inliquid phase. Binding was revealed using anti-HGF antibodies.Non-specific binding was measured by using BSA instead of purifiedreceptors in solid phase. (B, C, D) Binding of Decoy Met, Sema-PSI, andIPT to different forms of HGF. Engineered receptors were immobilized insolid phase and exposed to increasing concentrations of MYC-taggedactive HGF, pro-HGF, HGF-α or HGF NK1 in liquid phase. Binding wasrevealed using anti-MYC antibodies. Non-specific binding was measured byusing MYC-tagged angiostatin (AS) in liquid phase.

FIG. 3 shows that IPT domains 3 and 4 are sufficient to binding to HGF-αat high affinity. (A) Schematic representation of deleted IPT variants.Color code and legend as in FIG. 1A. (B) ELISA analysis of interactionsbetween IPT variants and HGF-α. Engineered IPTs were immobilized insolid phase and exposed to increasing concentrations of HGF-α in liquidphase. Binding was revealed using anti-HGF antibodies.

FIG. 4 shows that IPT domains 3 and 4 are sufficient for binding to HGFin living cells. (A) Schematic representation of the deleted MetΔ25-741receptor. Color code and legend as in FIG. 1A. (B) Surface biotinylationanalysis. Cellular proteins were immuno-precipitated (IP) usingantibodies directed against the C-terminal portion of Met and analyzedby Western blotting (WB) using horseradish peroxidase-conjugatedstreptavidin (SA). The same blots were re-probed with anti-Metantibodies. W.T., wild-type; A549, A549 human lung carcinoma cells; MDA,MDA-MB-435 human melanoma cells; TOV, TOV-112D human ovary carcinomacells; Empty V., empty vector. The p170 band corresponds to unprocessedMet; p145 is the mature form of the receptor. (C) Chemical cross-linkinganalysis. TOV-112D cells expressing Met_(Δ25-741) (Met Δ25-741) andwild-type TOV-112D cells (W.T. TOV) were incubated with HGF and thensubjected to chemical cross-linking. Cell lysates wereimmuno-precipitated using anti-Met antibodies and analyzed by Westernblotting using anti-HGF antibodies. Arrow indicates HGF-Met_(Δ25-741)complexes. (D) Met phosphorylation analysis. TOV-112D cells expressingMet_(Δ25-741) were stimulated with 1% FBS as a negative control and withequal amounts of HGF, pro-HGF, HGF NK1 or NK1-NK1. Receptorphosphorylation was determined by immuno-precipitation with anti-Metantibodies and Western blotting with anti-phosphotyrosine (anti-pTyr)antibodies. The same blots were re-probed using anti-Met antibodies.Arrows indicate bands corresponding to Met_(Δ25-741) or immunoglobulins(Ig). (E) Schematic representation of NK1-NK1. From N- to C-terminus:SP, signal peptide; ND, N-domain; K1, kringle 1; H, poly-histidine tag.

FIG. 5 shows that soluble IPT inhibits HGF-induced invasive growth invitro. (A) Lentiviral vector transduced MDA-MB-435 cells were stimulatedwith recombinant HGF and Met phosphorylation was determined byimmuno-blotting using anti-phosphotyrosine antibodies (upper panel). Thesame blot was re-probed using anti-Met antibodies (lower panel). EmptyV., Empty Vector. (B) Branching morphogenesis assay. Pre-formedspheroids of lentiviral vector-transduced MDA-MB-435 cells were embeddedin collagen and then stimulated with recombinant HGF to form branchedtubules. Collagen invasion was quantified by scoring the mean number oftubules sprouting from each spheroid. EV, Empty Vector; DM, Decoy Met;SP, Sema-PSI. (C) Representative images from the experiment described inB. Magnification: 200×.

FIG. 6 shows that soluble IPT displays anti-tumor and anti-metastaticactivity in mice. CD-1 nu−/− mice were injected subcutaneously withlentiviral vector-transduced MDA-MB-435 cells, and tumor growth wasmonitored over time. (A) Kaplan-Meier-like plots of tumor latency (Xaxis, time in days; Y axis, percent of tumor free-animals). Empty v.,empty vector. (B) Mean tumor volume over time. (C) Immuno-histochemicalanalysis of tumor sections using anti-FLAG antibodies. Magnification:400×. (D) Tumor vessel analysis. Tumor sections were stained withanti-Von Willebrand factor antibodies. The number of vessels per squaremm of tumor section was determined by microscopy. EV, Empty Vector; DM,Decoy Met; SP, Sema-PSI. (E) Metastasis incidence analysis. Uponautopsy, serial lung sections were analyzed by microscopy to determinethe presence of micrometastases. Metastasis incidence—i.e. the number ofmice with metastasis over the total—is indicated in both percentage(bars) and fraction (at the end of bars). (F) Representative images ofmicrometastases from the empty vector group. Lung sections were stainedwith hematoxylin and eosin. Dotted lines identify the walls of bloodvessels (vs). Metastatic cells (mc) can be found inside vessels as anembolus or in the parenchyma. Magnification: 400×.

The data presented in the present disclosure suggest that the α-chain ofHGF binds to the IPT region of Met at high affinity, and that it does soindependently of proteolytic processing of the ligand. They also suggestthat HGF binding to IPT in the context of a trans-membrane Met lackingthe Sema domain is sufficient for transmitting the signal for receptoractivation to the cytoplasmic kinase domain, although withoutdistinction between the inactive and active form of the ligand. Finally,they provide evidence that engineered proteins derived from the IPTregion and Sema domain of Met are capable of neutralizing thepro-invasive activity of HGF both in vitro and in vivo.

It has been known for long time that HGF is a bivalent factor. Earlyprotein engineering studies identified a high affinity Met-binding sitein the N domain and first kringle of HGF. Subsequently, combinedbiochemical and biological analysis demonstrated that the HGF serineprotease-like domain (β-chain), while not necessary for binding, plays akey role in mediating receptor activation. More recently, detailedcrystallographic and mutagenesis data have thoroughly characterized bothstructurally and functionally the low affinity Met-binding site on theβ-chain of HGF and its interaction with the Sema domain of Met. Theinterface between the α-chain of HGF and Met had remained howeverelusive. Small angle X-ray scattering and cryo-electron microscopystudies suggested the presence of contacts among the N-terminal andfirst kringle domain of HGF and the Sema domain of Met. However, plasmonresonance analysis revealed that this interaction has a very lowaffinity (about 2 times lower than that of HGF-β for Sema and 100 timeslower than that of HGF-α for the intact receptor). Since this weakinteraction cannot account per se for the tight bond between HGF andMet, the high affinity HGF-binding site on Met had still to beidentified.

The results presented here contribute to fill this gap and suggest thatthis long sought-after HGF-binding site lies in the IPT region of Metand more precisely in the last two immunoglobulin like domains close tothe cell membrane. Several distinct experimental evidences provided inthe present disclosure suggest that this is the case. Firstly, asoluble, deleted Met receptor containing nothing but the four IPTdomains (IPT) binds to HGF with substantially the same affinity as theentire extra-cellular portion of Met. Conversely, Sema displays very lowaffinity towards HGF. Secondly, IPT binds to active HGF, pro-HGF orHGF-α with unchanged strength. Thirdly, deletion of IPT 1 and IPT 2 doesnot affect the affinity of IPT for any form of HGF. Fourthly, anengineered Met receptor carrying a large deletion in its ectodomaincorresponding to the Sema domain, the PSI module and the first twoimmunoglobulin-like domains (Met_(Δ25-741)) retains the ability to bindto HGF and to transduce the signal for kinase activation to the insideof the cell, although it cannot distinguish between active HGF andPro-HGF. Finally, a dimeric form of HGF NK1, which is known to containthe minimal Met binding domain of HGF-α, is capable of elicitingactivation of Met_(Δ25-741) as efficiently as if not more powerfullythan HGF, thus identifying in IPT 3-4 the HGF NK1-binding site.

While these data point at a key role of IPT in HGF binding, it isnoteworthy that two previous structure/function studies on theextracellular portion of Met failed to identify any ligand binding sitein this region. A first draft of the Met ectodomain map suggested thatthe Sema domain is necessary and sufficient for HGF binding based onELISA assays. A second study analyzed the role of the Sema domain inreceptor dimerization and suggested that an engineered form of the Metextracellular portion containing a deletion in the Sema domain was notcapable of co-precipitating HGF.

The present disclosure further demonstrates that cooperation betweenSema and IPT is observed also when the extracellular portion of Met isused as a biotechnological tool to inhibit HGF-induced invasive growth.In the in vitro analysis and in mouse xenografts both the IPT andSema-PSI soluble proteins displayed a significant inhibitory effect.However, none of them could achieve the powerful inhibition displayed bythe full Met ectodomain, which contains both the low affinity and highaffinity HGF-binding site. This implies that both of these interactionscontribute to controlling Met activity. While the HGF-β-Sema contact hadalready been identified as a target for therapy, the results presentedhere unveil a second interface that offers opportunities forpharmacological intervention. Recombinant proteins or antibodies thatbind to the IPT region in place of bona fide HGF have an application ashighly competitive inhibitors of Met for the treatment ofHGF/Met-dependent cancers.

Materials and Methods

Protein Engineering

Soluble or trans-membrane receptors and engineered ligands described inthis work have been generated by standard PCR and genetic engineeringtechniques. All factors conserve the leader sequence of their parentalprotein at the N-terminus. The amino acid (aa) sequences of soluble Metproteins (Gene Bank N. X54559) correspond to aa 1-24 (signal peptide)plus: Decoy Met, aa 25-932; Sema, aa 25-515; Sema-PSI, aa 25-562;PSIIPT, aa 516-932; IPT, aa 563-932; IPT Δ1, aa 657-932; IPT Δ1-2, aa742-932; IPT-3, aa 742-838; IPT-4, aa 839-932. At the C-terminus of eachmolecule a double FLAG (SDYKDDDDK—SEQ ID No.:19) or single MYC(EQKLISEEDLN—SEQ ID No.:20) epitope sequence and a poly-histidine tag(HHHHHHH—SEQ ID No.:21) were added for protein detection andpurification. The transmembrane engineered MetΔ25-741 is identical towild-type Met except for the deleted region (aa 25-741). The amino acidsequences of engineered HGF proteins (Gene Bank N. M73239) correspond toaa 1-31 (signal peptide) plus: HGF, aa 32-728; HGF-α, aa 32-473; HGFNK1, aa 32-205; HGF-β, aa 495-728. The above MYC or FLAG epitope andpoly-histidine tag were added at the C-terminus. Uncleavable HGF hasbeen described before (Mazzone, M. et al. (2004) J Clin Invest. 114(10),1418-1432). NK1-NK1 is a dimeric form of HGF NK1 consisting of the sameN-terminal region HGF repeated in tandem (aa 1-205 directly linked to aa32-205 without spacer). The cDNAs encoding all engineered proteins weresubcloned into the lentiviral transfer vector pRRLsin.PPT.CMV.eGFP.Wpre(SEQ ID No.:1) in place of the gfp cDNA as disclosed in Follenzi, A. etal. (2000) Nat Genet. 25(2), 217-222. The GFP coding sequence wasreplaced by the following cDNAs: decoy met FLAG.his (SEQ ID No.:2), SemaFLAG.his (SEQ ID No.:3), Sema-PSI FLAG.his (SEQ ID No.:4), PSI-IPTFLAG.his (SEQ ID No.:5), IPT FLAG.his (SEQ ID No.:6), IPT Δ1 FLAG.his(SEQ ID No.:7), IPT Δ1-2 FLAG.his (SEQ ID No.:8), IPT 3 FLAG.his (SEQ IDNo.:9), IPT 4 FLAG.his (SEQ ID No.:10), Met Δ25-741 (SEQ ID No.:11), HGFMYC his (SEQ ID No.:12), HGF-α MYC his (SEQ ID No.:13), HGF-NK1 MYC his(SEQ ID No.:14), HGF-β MYC his (SEQ ID No.:15), uncleavable HGF MYC his(SEQ ID No.:16), NK1-NK1 his (SEQ ID No.:17), angiostatin MYC his (SEQID No.:18).

Enzyme-Linked Immunosorbant Assays

All engineered receptors and factors were collected from the conditionedmedium of lentiviral vector-transduced MDA-MB-345 human melanoma cellsin the absence of serum. Factor purification was performed byimmobilized-metal affinity chromatography as previously described inMichieli P. et al. (2002) Nat Biotechnol. 20(5), 488-495. Conversion ofpro-HGF into active HGF was performed by incubating purified pro-HGF(maximal concentration 100 ng/μl) with 2-10% FBS (Sigma, St. Louis, Mo.)at 37° C. for 24 hours. Factor conversion was analyzed by Westernblotting using anti-HGF antibodies (R&D Systems, Minneapolis, Minn.).Uncleavable HGF subjected to the same incubation with FBS was used aspro-HGF in all assays that compared active HGF with unprocessed HGF.Binding of engineered ligands to soluble receptors was measured by ELISAusing FLAG-tagged soluble receptors in solid phase and MYC-taggedengineered ligands in liquid phase. A fixed concentration of purifiedsoluble receptor (100 ng/well) was adsorbed to 96-well ELISA plates.Protein-coated plates were incubated with increasing concentrations ofengineered ligands, and binding was revealed using biotinylated anti-HGFantibodies (R&D Systems, Minneapolis, Minn.) or anti-MYC antibodies(Santa Cruz Biotechnology, Santa Cruz, Calif.). Binding data wereanalyzed and fit using Prism software (Graph Pad Software, San Diego,Calif.).

Cell Culture

MDA-MB-435 human melanoma cells were purchased from the GeorgetownUniversity Tissue Culture Shared Resource (Washington, D.C.). Cells weremaintained in DMEM supplemented with 10% FBS (Sigma). TOV-112D humanovarian carcinoma cells were obtained from ATCC (Rockville, Md.; ATCC N.CRL-11731) and were cultured using a 1:1 mixture of MCDB 105 Medium andMedium 199 supplemented with 15% FBS (all from Sigma). A549 human lungcarcinoma cells were also obtained from ATCC (ATCC N. CCL-185) andmaintained in RPMI supplemented with 10% FBS.

Lentiviral Vectors

Vector stocks were produced by transient transfection of 293T cells aspreviously described in Follenzi, A. et al. (2000) Nat Genet. 25 (2),217-222. Briefly, the plasmid DNA mix for transfection was prepared asfollows: ENV plasmid (VSV-G), 9 μg; PACKAGING plasmid pMDLg/pRRE 16.2μg; REV plasmid, 6.25 μg; TRANSFER VECTOR (plasmid #2-18), 37.5 μg. Theplasmids were diluted in a solution of TE/CaCl₂, to which a HBS solutionwas added while vortexing at maximum speed. The DNA/CaCl₂/HBS mix wasimmediately added drop-wise to the cell plates that were then incubatedat 37° C. After 14-16 hours the culture medium was replaced with a freshone. Cell culture supernatants containing vector particles werecollected about 36 hours after medium changing. After collection, thesupernatants were filtered through 0.2 μm pore membranes and stored at−80° C. Viral p24 antigen concentration was determined by the HIV-1 p24core profile ELISA kit (NEN Life Science Products, Boston, Mass.)according to the manufacturer's instructions. Cells were transduced insix-well plates (10⁵ cells/well in 2 ml of medium) using 40 ng/ml of p24in the presence of 8 μg/ml polybrene (Sigma) as described in Vigna, E.and Naldini, L. (2000) J Gene Med. 2(5), 308-316. Medium was changedabout 18 hours after transduction. Cell growth and protein productionwas monitored over time. Trandsuced cell lines were then seeded in 15 cmplates, grown to 80% confluence and incubated in medium without serum.After 72 hours, the supernatants containing the recombinant solubleproteins were collected, filtered and purified by affinitychromatography or stored at −30° C.

Immuno-Precipitation and Western Blot Analysis

Cell lysis, immuno-precipitation and Western blot analysis wereperformed using extraction buffer (EB) as described in Longati, P. etal. (1994) Oncogene 9(1), 49-57. Signal was detected using ECL system(Amersham Biosciences, Piscataway, N.J.) according to the manufacturer'sinstructions. Anti-Met antibodies for immunoprecipitation have beendescribed by Ruco, L. P. et al. (1996) J Pathol. 180(3), 266-270 andwere purchased from UBI (Lake Placid, N.Y.). Anti-Met antibodies forWestern blot were purchased from Santa Cruz. Anti-FLAG antibodies wereobtained from Sigma. Met phosphorylation analysis in lentiviral vectortransduced MDA-MB-435 cells was performed as previously described inMichieli, P. et al. (2004) Cancer Cell 6(1), 61-73.

HGF Cross-Linking and Met Activation Analysis

Lentiviral vector-transduced TOV-112D cells expressing Met_(Δ25-741)were subjected to surface biotinylation analysis using an ECL™ SurfaceBiotinylation Module kit (Amersham Biosciences) according to themanufacturer's instructions. Chemical cross-linking was performed aspreviously described in Mazzone, M. et al. (2004) J Clin Invest.114(10), 1418-1432. Briefly, cells were deprived of serum growth factorsfor 3 days and then incubated with 1 nM HGF for 3 hours. Cell lysateswere immuno-precipitated using antibodies directed against theC-terminal portion of Met as disclosed in Ruco, L. P. et al. (1996) JPathol. 180(3), 266-270, resolved by SDS-PAGE using a 3-10%polyacrylamide gradient and analyzed by Western blotting using anti-HGFantibodies (R&D). For receptor activation analysis, TOV-112D cellsexpressing Met_(Δ25-741) were deprived of serum growth factors for 3days and then stimulated with 1 nM HGF, Uncleavable HGF, HGF NK1 orNK1-NK1 for 10 minutes. Cells were lysed using EB as described inLongati, P. et al. (1994) Oncogene 9(1), 49-57. Cellular proteins wereimmuno-precipitated with anti-Met antibodies as above and analyzed byWestern blotting using anti-phosphotyrosine antibodies (UBI). The sameblots were re-probed with anti-Met antibodies (Ruco, L. P. et al. (1996)J Pathol. 180(3), 266-270).

Biological Assays

Collagen invasion assays using MDA-MB-435 cells were performed usingpreformed spheroids as described in Michieli, P. et al. (2004) CancerCell 6(1), 61-73. Briefly, spheroids were generated by incubating cellsovernight (700 cells/well) in non-adherent 96-well plates (Greiner,Frickenhausen, Germany) in the presence of 0.24 g/ml methylcellulose(Sigma). Spheroids were embedded into a collagen matrix containing 1.3mg/ml type I collagen from rat tail (BD Biosciences, Bedford, Mass.) and10% FBS using 96-well plates (40 spheroids/well). Embedded spheroidswere cultured at 37° C. for 24 hours, and then stimulated with 30 ng/mlHGF (R&D) or no factor for additional 24 hours. The number of tubulessprouting from each spheroid was scored by microscopy. At least 12spheroids per experimental point were analyzed.

Tumorigenesis Assays

Lentiviral vector-transduced MDA-MB-435 tumor cells (3·10⁶ cells/mouse)in 0.2 ml of DMEM were injected subcutaneously into the right posteriorflank of six-week old immunodeficient nu−/− female mice on Swiss CD-1background (Charles River Laboratories, Calco, Italy). Tumor size wasevaluated every 2 days using a caliper. Tumor volume was calculatedusing the formula V=4/3πx2y/2 where x is the minor tumor axis and y themajor tumor axis. A mass of 15 mm³—corresponding approximately to theinitial volume occupied by injected cells—was chosen as threshold fortumor positivity. Mice whose tumors were below this threshold wereconsidered tumor-free. After approximately 4 weeks, mice were euthanizedand tumors were extracted for analysis. Animals were subjected toautopsy. Tumors and lungs were embedded in paraffin and processed forhistology. Micrometastasis analysis was performed by microscopy onserial lung sections stained with hematoxylin and eosin. Tumor sectionswere stained with hematoxylin and eosin and analyzed by an independentpathologist not informed of sample identity. Transgene expression wasdetermined on tumor sections by immuno-histochemistry using anti-FLAGantibodies (Sigma). Sections were counterstained with Meyer hematoxylin(Sigma). Tumor angiogenesis was analyzed by immuno-histochemistry usinganti-Von Willebrand factor antibodies (DAKO, Glostrup, Denmark).Sections were counterstained as above. Vessel density was assessed bymicroscopy. At least 12 fields per animal were analyzed. All animalprocedures were approved by the Ethical Commission of the University ofTurin, Italy, and by the Italian Ministry of Health.

Statistical Analysis

Statistical significance was determined using a two-tail homoscedasticStudent's t-Test (array 1, control group; array 2, experimental group).For all data analyzed, a significance threshold of p<0.05 was assumed.In all figures, values are expressed as mean±standard deviation, andstatistical significance is indicated by a single (p<0.05) or double(p<0.01) asterisk.

Results

Engineering of HGF/Met Functional Domains

A schematic representation of the functional domains contained in Metand HGF is shown in FIG. 1A. The extracellular portion of Met includes aSema domain, a PSI hinge, and four IPT modules (left panel). HGF iscomposed of an α- and a β-chain joined by a disulphide bridge in themature protein. The α-chain in turn comprises an N-terminal domain andfour kringles (right panel). To analyze the interactions between Met andHGF, the inventors expressed all these functional domains as individual,soluble proteins. Functional domains were engineered to contain thesignal peptide of the parental protein at their N-terminus, so that theycould be properly secreted. At the C-terminus, an exogenous epitope(FLAG or MYC) for antibody recognition and a poly-histidine tag forprotein purification were added. All cDNAs encoding the engineeredfactors were subcloned into the lentiviral vector pRRLsin.PPT.CMV.Wpre,and recombinant lentiviral particles were produced as described inMaterials and Methods. Recombinant proteins were collected from theconditioned medium of lentiviral vector-transduced MDA-MB-435 humanmelanoma cells and purified to homogeneity by affinity chromatography.Purified proteins were quantified against standards by SDS-PAGE (FIG.1B).

ELISA Analysis of Met-HGF Interactions

The ability of Met ectodomains to interact with HGF was tested in ELISAbinding assays. Soluble receptors (Decoy Met, Sema-PSI, Sema, PSI-IPT,IPT) were immobilized in solid-phase and exposed to increasingconcentrations of active HGF. Binding was revealed using biotinylatedanti-HGF antibodies. Non-specific HGF binding was determined usingbovine serum albumin (BSA) in solid phase instead of soluble Metdomains. Binding affinity was determined by nonlinear regressionanalysis as described in Materials and Methods. In these conditions,decoy Met bound to HGF with a K_(D) of approximately 0.2-0.3 nM.Consistent with previous measurements, Sema-PSI and Sema bound to HGFwith an affinity at least one log lower compared to decoy Met.Surprisingly, both PSI-IPT and IPT bound to HGF very efficiently, withalmost the same affinity as decoy Met (FIG. 2A). The presence or absenceof the PSI domain did not affect the binding affinity for HGF of eitherSema or IPT. Since almost all Sema domains found so far in nature have aPSI module at their C-terminus, the inventors therefore continued thebinding analysis using decoy Met, Sema-PSI, and IPT.

To determine the affinity of each Met module for pro-HGF, HGF-α, HGF NK1and HGF-β and to compare it with that for active HGF, engineeredreceptors were immobilized in solid phase and exposed to increasingconcentrations of MYC-tagged ligands. Binding was revealed usinganti-MYC antibodies. Non-specific binding was determined using thekringle-containing protein angiostatin (AS)—also tagged with a MYCepitope—in liquid phase. Pro-HGF, HGF α-chain and HGF NK1, whichrepresents the minimal Met-binding module of HGF α-chain, bound to DecoyMet with a 3-, 4- and 10-time reduced affinity compared to active HGF,respectively (FIG. 2B). Binding of HGF-β to decoy Met (or to any otherMet domain) was too low to be detected in this kind of assay. Sema-PSIbound at a significant affinity to active HGF only, while binding topro-HGF, HGF-α or HGF NK1 was undistinguishable from non-specificbinding (FIG. 2C). In contrast, IPT bound to active HGF, pro-HGF andHGF-α with the same high affinity (FIG. 2D). HGF NK1 bound to IPT 10times less tightly than active HGF, i.e. with the same affinity as itbound to Decoy Met. These data suggest that the IPT region of Met bindsto the α-chain of HGF at high affinity independently of proteolyticprocessing of the ligand.

The α-Chain of HGF Binds to IPT Domains 3 and 4 with High Affinity

The IPT region of Met extends for about 400 amino acids and containsfour IPT domains. To more finely map the IPT-HGF interface, a series ofIPT variants that were deleted in one or more domains were engineered(FIG. 3A). IPT Δ1 and IPT Δ1-2 are two N-terminal deleted forms of IPTlacking the first or the first two immunoglobulin-like domains,respectively. IPT-3 and IPT-4 correspond to the two C-terminalimmunoglobulin-like domains expressed as single proteins. Proteinproduction and purification were performed as described above. Theability of the engineered IPTs to interact with HGF α-chain wasinvestigated in ELISA binding assays using the whole IPT region as acontrol. IPT, IPT Δ1, IPT Δ1-2, IPT-3 and IPT-4 were immobilized insolid phase and exposed to increasing concentrations of HGF-α. Bindingwas revealed using anti-HGF antibodies. Non-specific binding wasmeasured using BSA as above. As shown in FIG. 3B, deletion of the firsttwo immunoglobulin-like domains did not substantially affect HGFbinding.

In fact, IPT Δ1-2, a protein corresponding to the last twoimmunoglobulin-like domains of Met, bound to the α-chain of HGF withequal if not higher strength than IPT. However, further deletion ofeither the third or fourth immunoglobulin-like domain did almostcompletely impair HGF-αbinding. Similar results were obtained usingactive HGF or pro-HGF instead of HGF-α. These data suggest that the lasttwo immunoglobulin-like domains of Met, that lie close to thetrans-membrane helix in the context of a bona fide Met, are sufficientfor binding the α-chain of HGF at high affinity.

IPT Domains 3 and 4 are Sufficient for Binding to HGF in Living Cells.

To determine whether HGF could bind to IPT 3 and 4 in the context of amembrane-anchored receptor, a Met protein carrying a large deletion inthe extracellular region was engineered. Amino acids 25-741,corresponding to the Sema domain (aa 25-515), the PSI domain (aa516-562) and the first two IPT domains (IPT 1 and 2, aa 563-741) weredeleted, generating a recombinant receptor containing IPT domains 3 and4, the transmembrane helix and the full cytoplasmic region (FIG. 4A).The cDNA encoding the engineered receptor Met_(Δ25-741) was subclonedinto the same lentiviral vector described above. Recombinant lentiviralparticles were used to transduce the human ovary carcinoma cell lineTOV-112D, which lacks endogenous Met expression as determined by RT-PCRanalysis (Michieli, P., et al. (2004) Cancer Cell 6(1), 61-73). Surfacebiotinylation analysis revealed that Met_(Δ25-741) was properlyexpressed and exposed on the membrane of TOV-112D cells (FIG. 4B).

To examine whether Met_(Δ25-741) could bind to HGF, lentiviralvector-transduced cells were incubated in the presence or absence ofrecombinant HGF and subsequently treated them with the cross-linkingagent BS3. Cell lysates were immuno-precipitated with an antibody raisedagainst the C-terminal portion of Met, resolved by SDS PAGE and analyzedby Western blotting using anti-HGF biotinylated antibodies. As acontrol, the same analysis was performed on wildtype TOV-112D cells.Immunoblots showed a distinct band with a molecular weight ofapproximately 180 kD in the lane corresponding to cells expressingMet_(Δ25-741) treated with HGF but not in lanes corresponding to thesame cells without HGF or to wild-type TOV-112D cells, either in thepresence or absence of the ligand (FIG. 4C). Considering that bothMet_(Δ25-741) and HGF have a molecular weight of approximately 90 kDa,the immuno-precipitated cross-linked protein is compatible with acomplex formed by HGF plus Met_(Δ25-741).

HGF Binding to IPT Domains 3 and 4 Results in Met Activation in LivingCells.

The inventors next tested whether HGF binding to Met_(Δ25-741) couldinduce Met kinase activation. To this end, lentiviral vector transducedTOV-112D cells were stimulated with pro-HGF or active HGF, and celllysates were immunoprecipitated with anti-Met antibodies as above.Receptor activation was determined by Western blot analysis usinganti-phosphotyrosine antibodies. The same blots were re-probed withanti-Met antibodies to normalize the amount of receptorimmuno-precipitated. Remarkably, both pro-HGF and active HGF werecapable of inducing robust phosphorylation of Met_(Δ25-741) (FIG. 4D).Since pro-HGF binding to full-size Met does not induce kinaseactivation, this suggests that the Sema domain somehow exerts anauto-inhibitory effect on Met catalytic activity that is released uponbinding to active HGF. Receptor stimulation was also performed using HGFNK1 and an engineered dimeric ligand consisting of two NK1 fragmentsrepeated in tandem (NK1-NK1; FIG. 4E). As shown in FIG. 4D, NK1-NK1stimulation of lentiviral vector-transduced TOV-112D cells resulted inpotent phosphorylation of Met_(Δ25-741), while stimulation withmonomeric NK1 had no effect. These results suggest that the twoC-terminal IPT domains of Met (IPT 3 and 4) are sufficient to bind toHGF (and more precisely to HGF NK1 that represents the minimalMet-binding module in the α-chain of HGF) and to transmit the signal forreceptor activation to the cytoplasmic kinase domain, presumablyfollowing ligand-induced receptor dimerization. However, they alsosuggest that IPT 3 and 4 alone are not sufficient for distinguishing thebiologically active form of HGF from its inactive precursor, pro-HGF.

Soluble IPT Inhibits HGF-Induced Invasive Growth In Vitro.

In a previous study, it has been demonstrated that the extracellularportion of Met expressed as a soluble protein (Decoy Met) inhibitsHGF-induced invasive growth both in vitro and in mouse models of cancer(Michieli P. et al. (2004) Cancer Cell 6(1), 61-73). Recombinant solubleSema-PSI was also shown to inhibit both ligand-dependent and-independent Met phosphorylation (Kong-Beltran M. et al. (2004) CancerCell 6(1), 75-84). Based on these results, it has been tested whethersoluble IPT displayed HGF/Met antagonistic activity in living cells.MDA-MB-435 human melanoma cells, which express Met and are anestablished model system for analysis of HGF-mediated invasive growth,were transduced with lentiviral vectors encoding soluble Decoy Met,Sema-PSI or IPT. Cells transduced with an empty vector were used ascontrol. Lentiviral vector-transduced cells secreting comparable levelsof soluble factors (approximately 50 pmol/10⁶ cells/24 hours) wereserum-starved for several days, allowing the recombinant factors toaccumulate in the medium, and then stimulated with recombinant HGF. Mettyrosine phosphorylation was determined by immuno-blotting withanti-phosphotyrosine antibodies as described above. As shown in FIG. 5A,both IPT and Sema-PSI partially inhibited HGF-induced Metphosphorylation, while Decoy Met completely neutralized the ability ofHGF to induce Met activation. Re-probing of the same immuno-blots withantibodies directed against the C-terminal tail of Met revealed nosubstantial difference in the amounts of immuno-precipitated protein.

To test the inhibitory potential of Met ectodomains in a more biologicalsetting, the same cells were employed to perform an HGF-dependentbranching morphogenesis assay. Preformed cell spheroids were seeded in athree dimensional collagen matrix and then stimulated with recombinantHGF to form tubular structures. Branching was quantified by scoring themean number of tubules sprouting from each colony. As shown in FIG. 5B,both soluble IPT and Sema-PSI inhibited HGF-induced colony branching(Empty vector, 17.5 tubules/colony; IPT, 4.0 tubules/colony; Sema-PSI,6.7 tubules/colony). However, consistent with the results obtained inphosphorylation experiments, decoy Met was a more potent HGF-inhibitorthan either of its subdomain (2.5 tubules/colony). Representative imagesof colony morphology are shown in FIG. 5C.

Soluble IPT Displays Anti-Tumor and Anti-Metastatic Activity in Mice.

The above results prompted the present inventors to explore thetherapeutic potential of soluble IPT in mouse models of cancer.Lentiviral vector-transduced MDA-MB-435 melanoma cells were injectedsubcutaneosly into CD-1 nu−/− mice, and tumor growth was monitored overtime. After approximately three weeks, tumors were extracted foranalysis, and mice were subjected to autopsy. In a Kaplan-Meier-likeanalysis, where the percentage of tumor-free animals is plotted againsttime and tumor latency is quantified calculating the median in days, allengineered soluble receptors delayed the appearance of experimentaltumors. However, IPT was slightly more effective than Sema-PSI and decoyMet was more potent than either IPT or Sema-PSI (FIG. 6A). Analysis oftumor burden over time revealed that IPT was only slightly lesseffective than decoy Met, while Sema-PSI inhibited neoplastic growthonly during the very early stages of the experiment (FIG. 6B).Immuno-histochemical analysis of transgene expression showed that DecoyMet, Sema-PSI and IPT reached similar levels and distribution in tumors(FIG. 6C).

As HGF is a potent pro-angiogenic factor, it has been determined whetherinhibition of HGF/Met in tumors resulted in impairment of angiogenesis.Tumor sections were analyzed by immuno-histochemistry using antibodiesagainst Von Willebrand factor, and vessel density was assessed bymicroscopy (FIG. 6D). IPT decreased tumor vessel density by 1.5 times,while decoy Met achieved a much stronger inhibition (approximately 4times); Sema-PSI did not significantly affect tumor angiogenesis. Uponautopsy, lungs from the mice described above were extracted andprocessed for histology. Serial lung sections were stained withhematoxylin and eosin, and analyzed by microscopy to determine thepresence of micrometastases. The results are shown in FIG. 6A. In thecontrol group, 4 out of 6 mice (67%) were bearing micrometastases. Inthe IPT and Sema-PSI group, micrometastases could be found in only 1 outof 6 mice (17%), while no metastasis could be found in the decoy Metgroup. Metastatic lesions were both parenchymal (extravascular) andembolic (intravascular; see FIG. 6F for representative images).

The identification of the high affinity HGF binding site on the HGFRprovided in the present disclosure allows designing of novel proceduresleading to the generation of more specific inhibitors/antagonists of HGFand of HGFR. The following are non-limiting examples of novel methodsfor the identification of inhibitors/antagonists of HGF/HGFR targetingthe high affinity binding site of HGFR or utilizing the high affinitybinding site of HGFR as a tool to generate novel inhibitors/antagonists.

Development of a Monoclonal Antibody that Binds to Extracellular IPT-3and IPT-4 Domains of HGFR Preventing HGF Binding

Given that interaction of HGF with the extracellular IPT-3 and IPT-4domains is essential for high affinity HGF binding, one can generatespecific monoclonal antibodies that bind to IPT-3 and IPT-4 and competewith HGF for HGFR binding. This can be achieved by several strategies.

(A) A recombinant protein or peptide derived from IPT-3 and IPT-4 isgenerated by standard genetic engineering technology or chemicalsynthesis. This protein or peptide is injected into an appropriatelaboratory animal (usually a mouse or a rat) to give rise to an immunereaction. Splenocytes are then isolated from the immunized animal andfused to a myeloma cell line, and antibody-producing hybridoma clonesare selected by standard monoclonal antibody technology. Antibodiesdirected against IPT-3 and IPT-4 are then screened by an ELISA methodsimilar to the ones described in this disclosure that utilizesrecombinant IPT-3 and IPT-4 in solid phase and hybridoma-producedantibodies in liquid phase. Binding is revealed using anti-mouseimmuno-globulin antibodies that are available commercially.Alternatively, antibodies are screened for their ability to displacerecombinant HGF (in liquid phase) from IPT-3 and IPT-4 (in solid phase),or for their ability to immuno-precipitate recombinant IPT-3 and IPT-4proteins.

(B) A polynucleotide sequence coding for IPT-3 and IPT-4 inserted in anappropriate expression vector is injected directly into a laboratoryanimal to give rise to an immune response against the gene product.Antibodies directed against IPT-3 and IPT-4 are then isolated andscreened as described above.

(C) A polynucleotide sequence coding for IPT-3 and IPT-4 inserted in anappropriate expression vector is transferred into a mammalian cell linenot expressing HGFR to obtain expression of IPT-3 and IPT-4 on the cellsurface. Cells expressing IPT-3 and IPT-4 are then injected into alaboratory animal to give rise to an immune response, and antibodiesdirected against IPT-3 and IPT-4 are isolated and screened as describedabove.

(D) A library of native antibodies generated by standard geneticengineering techniques (for example by the technology known as phagedisplay) from mammalian lymphocytes (preferably human, for example fromlymphocytes infiltrating a tumor expressing HGFR) is screened usingrecombinant IPT-3 and IPT-4 proteins. Positive clones (i.e. those clonesthat bind to IPT-3 and IPT-4 at high affinity) are then isolated,expanded, and the antibody characterized biochemically.

(E) Human memory B cells are isolated from the peripheral blood of apatient harboring a tumor expressing HGFR as disclosed by severalstudies including Traggiai E. et al. (2004) Nat Med. 10(8), 871-875.Once cultures of immortalized memory B cells are established, a skilledartisan in the field can screen for cells secreting an antibody directedagainst IPT-3 and IPT-4 using the methods described here above in (A).Having identified such antibody-producing cells, the desired antibodycan be cloned by polymerase chain reaction and standard geneticengineering procedures.

Identification of a Test Compound that Binds to Extracellular IPT-3 andIPT-4 Domains of HGFR Inhibiting HGFR Activity

With a different approach, it is possible to isolate test compounds ofdiverse origin that bind to the high affinity HGF binding site of HGFRand interfere with HGF-induced HGFR activation. This can be achieved byseveral strategies.

(A) Using ELISA assays similar to those described in this disclosure, askilled artisan in the field can screen a compound library (includingbut not limited to a synthetic chemical library, a natural compoundlibrary, a small molecule library, a peptide library) for agents thatdisplace HGF interaction with IPT-3 and IPT-4. In this kind of assays,recombinant IPT-3 and IPT-4 protein is immobilized in solid phase andincubated with a fixed amount of HGF in liquid phase. Following exposureto library compounds, HGF binding is measured with commerciallyavailable anti-HGF antibodies.

(B) Using a cell line expressing an engineered form of the HGFRcontaining nothing but the IPT-3 and IPT-4 domains in the extracellularpart similar to that described in this disclosure (Met_(Δ25-741)), askilled artisan in the field can screen a compound library (includingbut not limited to a synthetic chemical library, a natural compoundlibrary, a small molecule library, a peptide library) for agents thatdisplace HGF interaction with IPT-3 and IPT-4 or inhibit HGF-inducedHGFR activation. This can be achieved by putting said engineered cellsin contact with the library and then measuring HGF-induced HGFRphosphorylation as described in the present study, or by other meansthat reveal HGFR activation including a scatter assay, a reconstitutedmatrix invasion assay, a branching morphogenesis assay, a cell survivalassay or other in vitro biological tests as described in Michieli, P. etal. (2004) Cancer Cell 6(1), 61-73.

(C) Using the engineered cell line described above, a skilled artisan inthe field can screen a genetic library (including but not limited to acDNA expression library, a short hairpin RNA library, an antisense DNAlibrary, a random nucleotide library) for polynucleotides or geneproducts that displace HGF interaction with IPT-3 and IPT-4 or inhibitHGF-induced HGFR activation. This can be achieved by transfecting,transducing or anyway introducing the nucleotide library into said cellsand then testing the ability of HGF to activate the deleted form of HGFRexpressed by the same cells. HGFR activation is measured as described in(B).

Exemplary Functional Assays that Measure the Biological Activity ofCompounds that Bind to Extracellular IPT-3 and IPT-4 Domains of HGFR

Whatever strategy is employed to generate anti-IPT antibodies orIPT-binding compounds, the final product (i.e. monoclonal antibodiesdirected against IPT-3 and IPT-4 or natural or synthetic compounds thatbind to IPT-3 and IPT-4) is then subjected to biological assays aimed atdetermining whether these agents have the ability to interfere with HGFRactivity. These assays can be performed in vitro using culturedmammalian cells or in vivo using laboratory animals.

(A) Scatter assay. Epithelial cells growing in compact colonies in aPetri dish and expressing HGFR are induced to ‘scatter’ by stimulationwith HGF. As a result of HGF stimulation, cells in the Petri dish appearmore separated and dispersed. This assay can be performed in thepresence of several test compounds. Among the compounds tested, anHGF/HGFR inhibitor/antagonist can be identified by the absence of ascattered phenotype in response to HGF stimulation.

(B) Cell migration assay. Cells expressing HGFR have the ability tomigrate towards an HGF gradient. In other words, cells are attracted bychemotaxis towards higher concentrations of HGF. This ability can beexploited to screen for HGF inhibitors in a Boyden chamber assay. Cellsare seeded in the first chamber and HGF is applied in the second chamberthat is separated from the first by a porous membrane. Cells migratethrough the membrane and reach the second chamber were HGF is moreconcentrated. Agents that inhibit this process are identified asHGF/HGFR inhibitors/antagonists.

(C) Transwell™ migration assay or reconstituted matrix invasion assay.This is a variation of the assay described in (B) in which the porousmembrane is covered with a layer of collagen, Matrigel™, or otherreconstituted organic matrices. Cells have to digest the organic matrixin order to migrate through it. This is a more rigorous assay thatmeasures invasion rather than simple migration of cells.

(D) Collagen invasion assay or branching morphogenesis assay. In thisassay, mammalian cells expressing HGFR (preferably epithelial cells orcarcinoma cells) are seeded in a tridimensional layer of collagen andthen allowed to grow until they form spheroids of approximately 1,000cells each. Alternatively, spheroids can be pre-formed ahead byincubating cells overnight in non-adherent 96-well plates in thepresence of methylcellulose as disclosed in Michieli, P. et al. (2004)Cancer Cell 6(1), 61-73. Once spheroids are embedded in the collagenlayer, they are stimulated with HGF and incubated at 37° C. This resultsin the sprouting of hollow tubules from the spheroids; each tubule isformed by several cells organized in a tubular structure and polarizedso that there is a side of the cell that sees the lumen and a secondside that sees the medium outside. As the assay goes on tubules tend tobranch and to form a more complex architecture. This assay is highlyspecific for HGF. Agents that inhibit this process have a highprobability to represent very specific HGF/HGFR antagonists.

(E) Mitogenic assay. HGF has the ability to induce DNA replication andcell division in some cell expressing HGFR. The most responsive cellsare certainly primary hepatocytes, usually mouse or rat. To testHGF-induced DNA replication, cells are deprived of serum growth factorsand then stimulated with increasing concentrations of HGF. Shortlyafter, radioactive thymidine is added and the cells are incubated at 37°C. for approximately one day. Following extensive washing and fixation,radioactive thymidine incorporated into cell DNA is measured by liquidscintillation counting or other standard methods that allow radiationquantification.

(F) Survival assay. HGF has the ability to protect HGFR-expressing cellsagainst apoptosis or programmed cell death. This can be exploited tomeasure HGFR activity in a survival assay. Cells are pre-incubated withHGF and the test compound (potential HGFR inhibitor), and then subjectedto an apoptotic stimulus such as a toxic drug, absence of adherence,hypoxia, heat shock, radiation, or DNA damage. After an appropriate timeinterval, cell death is measured by standard methods including TUNEL(Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling),nucleosomes, DNA ladder, caspase activity, vital dye staining or anyvariation thereof.

(G) Mouse tumorigenesis assay. In this kind of assay, the activity of apotential HGF/HGFR inhibitor is tested directly in a laboratory animal,preferably a mouse or a rat. There are several methods for obtaining atumor in a mouse. The most utilized strategy is to create a xenograft,i.e. to transplant tumor cells (usually of human origin) into an animalrecipient (usually an immunodeficient mouse). Cells can be implantedsubcutaneously (a quick and simple method to obtain an experimentaltumor) or orthotopically, i.e. in the same organ where the tumor cellhas been isolated (e.g. a breast carcinoma into the mammary fat pad, acolon carcinoma into the intestine mucosa, a hepatocarcinoma into theliver parenchyma and so on). Whatever the method employed is, injectionof tumor cells into a laboratory animal gives rise to an experimentaltumor. This tumor-bearing animal can now be used to evaluate theanti-tumor potential of test compounds. Anti-HGF/HGFR antibodies orcompounds can be delivered to an animal carrying a tumor lesion withdysregulated HGF/HGFR signaling by the most appropriate existing methodincluding intravenous injection, intraperitoneal injection, osmoticpump, oral administration, suppository, gene therapy protocol, localadministration and so on. After an appropriate treatment period, theanimal is euthanized and its tumor and organs are explanted foranalysis.

(H) Mouse metastogenesis assay. Experimental metastases can be inducedin a mouse by systemic injection of tumor cells. These get entrapped inthe lung capillaries and subsequently extravasate to give rise topulmonary metastases. These can be measured upon autopsy by severalapproaches including microscopy, histological analysis,immuno-histochemical methods, whole-body imaging. The test compounds aredelivered as described in (G).

(I) Gene therapy protocol. In case the HGF/HGFR inhibitor is anantibody, a recombinant protein, a peptide or a small interfering RNA,delivery to the tumor-bearing animal can be achieved by a gene therapyapproach. This consists in introducing the desired polynucleotide intoan appropriate delivery vector that can be chosen among lentiviralvectors, adenoviral vectors, retroviral vectors, naked DNA, or anyvariation thereof. The vector preparation can be delivered sytemicallyor locally to the tumor depending on the tumor site, on the vector or onthe tumor histotype. The biological effects of gene therapy are analyzedas described for other compounds in (G).

1. Vector comprising at least a nucleotide sequence encoding theextra-cellular IPT-3 and IPT-4 domains of hepatocyte growth factorreceptor for use as a medicament for treating cancer.